Persistent pulmonary hypertension of the newborn (PPHN) is a syndrome that is characterized by failure of the pulmonary circulation to achieve or sustain the normal decrease in pulmonary vascular resistance (PVR) at birth. Mechanisms that contribute to high PVR in PPHN are uncertain, but include abnormalities of vascular function and structure, and in severe cases, altered lung growth (PPHN with lung hypoplasia). Past studies have shown that endothelial dysfunction, including impaired production of vasodilators, such as nitric oxide (NO), contributes to abnormal vascular tone and reactivity in an experimental model of PPHN. However, mechanisms that regulate endothelial cell growth and function during normal lung development, or that impair endothelial function in PPHN are unclear. In addition, mechanisms that impair lung angiogenesis in the setting of severe PPHN are unknown. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and critical trophic factor for maintaining normal endothelium. Past studies of genetic mouse models of development have shown that VEGF is essential for vasculogenesis during the early embryonic period, but less is known about the role of VEGF later during development, especially in the pulmonary circulation. The endothelial cell plays a central role in regulation of vascular tone in the normal perinatal lung, especially through production of NO. NO modulates basal PVR in the normal fetus, contributes to the fall in PVR at birth, and is decreased in an experimental sheep model of PPHN. In vitro studies suggest that VEGF upregulates endothelial NO synthase (eNOS) expression, and that VEGF mediates angiogenesis through NO-dependent mechanisms in diverse settings. In this grant period, we have shown that lung VEGF expression is markedly decreased in experimental PPHN, and that chronic inhibition of VEGF in normal fetal sheep impairs endothelium-dependent vasodilation, downregulates eNOS, and causes severe pulmonary hypertension. In addition, we have recently found that pulmonary hypertension induced during early lung development decreases angiogenesis and impairs alveolarization in fetal sheep, providing a model of severe PPHN with lung hypoplasia. Based on strong preliminary data, we hypothesize that disruption of VEGF signaling prior to birth impairs endothelial function, causing severe pulmonary hypertension, altered vasoreactivity, and decreased angiogenesis in severe PPHN. In this grant renewal, we now propose to determine whether early disruption of VEGF and NO signaling inhibits angiogenesis and causes severe PPHN with lung hypoplasia and whether novel treatment strategies that augment VEGF and NO signaling can preserve endothelial survival and function, improve pulmonary hypertension and enhance angiogenesis in PPHN.